About Gene Therapy
Over the last few years, gene therapy (GT) has emerged as a promising medical tool to treat diseases. This novel approach is underpinned by the positive clinical results achieved in patients followed by regulatory authority approvals for in vivo product commercialization in both the USA and Europe; recently for Luxurna™ (Spark Therapeutics Incorporation) by the US Food and Drug Administration (FDA) in 2017 and more recently by the European Medical Agency (EMA) in 2018, followed by Tegsedi™ (Ionis Pharmaceutical and Akcea Therapeutics) and Onpattro™ (Alnylam Pharmaceuticals) in 2018. These milestones have been considered as breakthroughs in the medical field and have encouraged pharmaceutical companies to increase investments in gene therapy product development and expansion of manufacturing capacities to support development and commercial supply. In 2018 FDA received 206 GT relevant IND submissions (Presented at the American Society of Gene & Cell Therapy conference 2019 by Raj Puri, MD, PhD, FDA/CBER, Silver Spring, MD presentation: “FDA Regulation of Cell & Gene Therapies: Facilitating Advanced Manufacturing”) and the FDA anticipates it will approve ten to twenty cell and gene therapy products a year by 2025. The recent gene therapy asset acquisitions of Spark Therapeutics by Roche and Brammer by ThermoFisher confirm the changes in product development strategy.
In vivo gene therapy applications utilize gene replacement to treat monogenic diseases, while targeting specific organs or cells (e.g. liver, muscles, CNS). For this purpose, different gene delivery systems such as viral (Retroviruses, Adenoviruses, Lentiviruses, Adeno-Associated Viruses, etc.) and non-viral vectors (naked DNA, cationic liposomes, etc.) have been developed in the last decades and used in clinical trials for a broad range gene therapy applications.
What is the Vehicle of Choice?
Different delivery strategies are under investigation, each having limitations and no vector can be universally applied for all indications. During the last two decades, Adenoviruses have been the most preferred gene delivery system in clinical trials. However, their preparation and application in patients have shown several major drawbacks, the most concerning is high immunogenicity observed in a broad range of patients.
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Therefore, the use of other vectors as a suitable alternative to Adenoviruses in clinical trials is gaining importance. Among the different available viral vectors and according to the clinical data provided by Wiley for gene therapy trials worldwide, Adeno-Associate Viruses (AAVs) and Lentivirus programs are gaining importance fostered by their low pathogenicity and ability to infect dividing and non-dividing cells. AAVs show broad tropism being able to target different organs, depending on their serotype, while lentiviruses seem to have more application ex vivo in Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T).
Accelerated Product Delivery for Tox and for Clinical Trials
Providing sufficient clinical material according to clinical timelines and in a cost-effective way is a significant challenge. Developing routine commercial manufacturing at a reasonable cost of goods is one of the major hurdles for successful development of gene therapy products. Using a flexible production platform for gene therapy products, significantly accelerates time to clinic and allows rapid supply of a broad range of different drug substances. Takeda’s Gene Therapy Center Austria (GTCA) has already established a versatile AAV platform suitable for many serotypes, which minimizes efforts needed for process development, scale-up and tech transfer and derisks clinical supply.
However, the production of viral vectors is complex and not very effective, compared to recombinant proteins, yields are low and purity is moderate. Viral vector manufacturing processes have to be significantly improved to meet rising demands and increasing quality requirements.
Scalability, process robustness and yield are the main parameters for successful upstream development. High titers in a bioreactor rely mainly on an appropriate host cell line (optimized for the production of viral particles), the cell culture media and the vector characteristics as well as the transfection approach, followed by other standard process-derived parameters such as dissolved oxygen, temperature, etc.
For downstream processing isolation of viral particles and reduction of process- and product-related impurities needs to be achieved while preserving potency and yield targets. The majority of industrial separation methods and chromatographic resins were designed for small molecules and recombinant proteins not considering virus specific requirements.
Increasing demands of viral vectors for gene and cell therapy at reasonable cost of goods can only be met by implementation of novel technologies and development of viral vector specific separation approaches.
Different Vectors, Different Purification Challenges
A production process should be robust, scalable, cost-effective and ideally applicable for different gene therapy products. Nevertheless, due to the variety of the innate molecule properties, tailored process adaptations are essential to fulfill quality requirements and yield targets. The highest costs in such a production process arise from the downstream processing, therefore understanding the biophysical properties of the viral vectors (e.g. stability, isoelectric point, etc.) helps to choose the optimal parameters and conditions and subsequently, can be beneficial to decide which unit operations or process steps can be reduced to minimize product losses during the process.
The most common downstream unit operations used for a gene therapy production process include a clarification step to separate the cells or cell debris from viral particles; ultra- and diafiltration for reducing the volumes and concentrating the product, different chromatography approaches to capture and polish the product and virus inactivation-dedicated steps for biosafety purposes.
Adeno-Associate Viruses
Different AAV serotypes are used for clinical studies depending on the targeted cell types and require subtype specific purification approaches to achieve high recovery yields while maintaining product potency and integrity.
The separation of cells from product is usually the last step of an upstream process but significantly impacts on the subsequent downstream step. The clarification step needs to be designed to remove all cells and cell debris without reducing the amount of viral particles in the clarified supernatant. Non-specific adsorption of viral particles on membranes and surfaces and high shear forces with negative impact on viral particle integrity are decreasing virus recovery. Proper screening of membrane types and buffer conditions for each viral vector is needed to optimize recovery, purity and potency.
An additional point to consider is how and if the viral particles are released from the host cells. Some cell lines release the majority of produced AAV particles during fermentation without cell lysis, simplifying the recovery. However, it is generally known that some AAV capsids tend to remain in the host cell so that a cell disruption process is needed to get access to the product. In other cases, AAV particles stick to the cell debris, cell membranes and/or host cell impurities leading to low yields or insufficient removal of process related impurities and out of specification results.
Chemical or mechanical processes need to be used for cell disruption in case of insufficient viral particle release increasing the amount of impurities and requiring substantial process development to meet yield and purity targets. Key parameters to optimize include conductivity, pH, temperature and process time. Commercially available products, such as Benzonase, are currently used for enzymatic digestion and removal of cellular and viral nucleic acid.
However, increased impurities during the harvest procedure can lead to premature filter clogging (membrane fouling) and subsequently, to increased retention pressures in the system facilitating the release of additional host proteins and genomic DNA from the cells into the retentate. Furthermore, hauling these additional impurities along a chromatography step can result in rapid resin fouling and poor resolution.
For protein purification, chromatography based on resins is the most preferred system because of its robustness and high selectivity. Membrane absorbers offer an alternative option to the conventional beads and allow for high flow rates resulting in reduced process time. Application of mild process conditions in combination with short contact time preserve biopotency. However, there is still a limited portfolio offered by suppliers and their use for gene therapy products has to be further evaluated.
The use of appropriate ligands specific to viral vectors and suitable for large scale processes (e.g. affinity chromatography) can be beneficial to reduce the number of purification steps. However, only a few affinity ligands are currently available for capturing AAV particles. The process-related conditions for each serotype and specific resin regeneration protocols have to be optimized case-by-case. This strategy is already implemented in Takeda’s AAV purification toolbox.
Another essential aspect to keep in mind is the full/empty ratio of final AAV drug substance. There is no standardized full/empty ratio in the industry for dosing patients with AAV and it differs from company to company but increasing the percentage of full capsids may be advantageous when reduced volumes are needed to treat specific diseases, e.g applications in central nerve system (CNS). The use of a drug substance with a high amount of empty capsids would require an increase of dose applied to the patients leading to unnecessary stress to the immune system.
Presently there are only a few suppliers with commercially available chromatographic resins able to separate full from empty capsids. Monolithic columns can be used for different AAV serotypes. The complexity of this separation relies on the close biophysical properties between full and empty capsids. Nevertheless, although these resins (e.g. Monoliths) achieve an acceptable separation full/empty ratio, further eff orts have to be made to improve purification protocols in order to increase yields.
Ultracentrifugation can be used as an alternative in order to achieve full/empty separation. This approach relies on highly qualified operators and brings some complexity for routine production and scale-up. Nevertheless, Takeda’s Gene Therapy Center Austria has successfully implemented a proprietary scalable and reproducible approach based on an automated ultracentrifugation step running as a close system in Good Manufacturing Practice (GMP) production plant (Figure 1).
Lentivirus
Lentivirus is a class of retrovirus with the ability to transfer genetic material to dividing and non-dividing cells. The downstream process does not require a full/empty ratio separation. Nevertheless because of its lipid shell, stability and potency properties can be affected when a purification process is not adequate. Another aspect to consider is the fact that most lentivirus processes were established in adherent cells, which make scalability in the upstream process as well as full recovery more difficult.
The purification of LV can be challenging due to its high sensitivity to shear stress, temperature and pressure. These conditions can be given during a standard filtration, a chromatographic step or any chemical process step used to remove impurities. The use of an appropriate DNA removal step and any other impurity clarification step to reduce hauling impurities along the subsequent purification steps, facilitates for instance the regeneration of chromatography resins used in the process and avoids premature fouling. In addition, the use of nonstandardized chromatography resins shows poor loading capacities.
Critical Impurities Throughout the Process
Irrespective of the viral system used, the gene therapy approach is still relatively immature and not all safety relevant aspects and long-term implications are understood. Manufacturing processes for viral vectors are more complex compared to recombinant proteins and substantial process development is needed to improve process robustness, scalability and productivity. Due to the complexity of viral vectors, product characterization is challenging and new methods need to be developed to fully characterize gene therapy products and process impurities.
Regarding AAV, many efforts are undertaken for the characterization of full particles packaged with the “wrong” encapsidated DNA. Although there is no standardized method for the analysis of these contaminants, the use of Next Generation Sequencing (NGS) is gaining importance in the community. Indeed, these AAV particles may contain traces of backbone and helper plasmids (both used during the transfection) and even host cell line DNA might be packaged. The presence of such particles in the final product represents some concerns from a biosafety perspective for the patients: Immune response due to the presence of prokaryotic sequences; transfer of antibiotic resistance genes; human oncogenes (E1, E4, cellular genes); viral genes from Adenoviruses; and also may reduce the therapeutic biopotency.
During the last years, qPCR was used for the quantification of AAV vector genome and calculation of AAV full/empty ratio for patient dosing. Nevertheless, the high variability of results in samples with complex backgrounds has led to the implementation of alternative methods such as Digital Droplet PCR. This improved technology provides an absolute nucleic acid quantification with enhanced resolution.
However, there are still some limitations with PCR and for biosafety reasons, it is crucial to be able to calculate an accurate percentage of AAV full/empty capsids and demonstrate drug substance consistency from lot-to-lot. Takeda’s Gene Therapy Center Austria has implemented a more accurate methodology to determine AAV full/empty ratio. Their innovative methodology uses a combination of ELISA and CryoTEM Analysis for dosing patients in clinical trials Phase I/II and was already approved and acknowledged by the regulatory authorities.
Other analytical methods for AAV product characterization are well established and standardized. The most popular include ELISA, SDSPAGE, HPLC and Mass Spectroscopy. These methods are used for monitoring process-related impurities, which may derive from the standard materials used for the vector production and include cell culture media components, transfection reagents (plasmids and polyethylenimine), residual host-cell DNAs and proteins, and the reagents used for Solvent/Detergent treatment for viral clearance. Other product-related impurities innate to the AAV particle include vector aggregates and fragments, empty capsid particles (for AAV), co-packaged/random packaged host-cell and plasmid-derived DNA.
Process and product characterization need to be considered over the course of product development and need to be specifically addressed during process validation, process optimization and tech transfer.
Summary
In the areas of gene and cell therapy, more than 450 substances are currently in clinical trials and the number of approved products is expected to grow rapidly over the next years. Those products are very complex and require innovative approaches to meet safety requirements, clinical and market demands and cost of goods targets. Smart combinations of traditional downstream approaches and novel technologies are needed to develop scalable and robust purification processes for cell and gene therapy products.
References
- Nayerossadat N, Maedeh T, Ali PA. Viral and nonviral delivery systems for gene delivery. Adv Biomed Res. 2012;1:27. doi:10.4103/2277-9175.98152
- Ankita Desai & Philipp Nold. Cell & Gene Therapy Insights 2019; 5(3), 375-381.
- Pettitt, David. (2017). Scalable Purification of Viral Vectors for Gene Therapy: An Appraisal of Downstream Processing Approaches, BioProcess International 2017.
- Terova, Orjana & Soltys, Stephen & Hermans, Pim & De Rooij, Jessica & Detmers, Frank. Overcoming Downstream Purification Challenges for Viral Vector Manufacturing: Enabling Advancement of Gene Therapies in the Clinic. Cell and Gene Therapy Insights. 4. 101-111. 10.18609/cgti.2018.017.
- Halioua-Haubold CL, Peyer JG, Smith JA, et al. Regulatory Considerations for Gene Therapy Products in the US, EU, and Japan. Yale J Biol Med. 2017;90(4):683–693. 2017 Dec 19.
- Molecular Therapy — Methods & Clinical Development (2016) 3, 16002; doi:10.103838/mtm.2016
Author Biography
Dr. Juan A. Hernandez Bort works at Takeda - Gene Therapy Center Austria as Head of Gene Therapy Technologies and he collected, in the past 20 years, extended experience in the upstream and downstream process development of plasmaderived products, recombinants and lately gene therapy products. He holds a Master Degree and PhD in Biotechnology from the University of Natural Resources and Life Sciences in Vienna (BOKU), Austria. His current work is focused on Gene Therapy Process Development.